Project Summary Perturbation to the human gut microbial community is associated with various gastro-intestinal diseases and metabolic ill-health, especially obesity, but the complexity of this system is hampering the development of reliable microbiological therapies, e.g. probiotics. The naturally simple gut microbial community in Drosophila fruit flies offers a superb model for the overall research goal to determine the mechanistic basis of interactions between gut microbiota and host metabolic health. Building on evidence that the gut microbiota protects Drosophila against hyperlipidemia and hyperglycemia, the first aim of this project is to identify the microbial fermentation products of gut microbiota that protect the host against excessive energy storage (i.e. high levels of lipid, glycogen and sugars), by identifying the metabolites released from microbial communities that reduce energy storage indices; these functional metabolites likely include the products of polymicrobial metabolism. The second aim will test the hypothesis (based on preliminary data) that microbial-mediated reduction in energy storage is promoted by enhanced mobilization of free sugar, lipid and glycogen in the gut epithelium. The metabolic response of the host to the gut microbiota and their metabolic products will be quantified by isotopic analysis of dietary glucose metabolism in flies colonized with different microorganisms, and the role of candidate metabolic enzymes in reducing energy storage will be identified by RNAi-expression knockdown in the fly gut. These data will inform the third aim, to establish the mechanistic basis of the efficacy of probiotic Lactobacillus to reduce energy storage in flies associated with unmanipulated microbiotas that vary in capacity to reduce energy storage. Using metabolic modeling informed by transcriptomic and metabolomic data, the metabolic reactions and pathways (in the host and microbiota) that mediate microbiota-mediated reduction in energy storage will be identified; and the contribution of metabolic function, abundance and persistence of orally-administered Lactobacillus in the gut to probiotic-mediated protection against hyperglycemia and hyperlipidemia will be determined. Overall, this project will identify microbial products and host metabolic responses that underpin gut microbiota-mediated protection against hyperglycemia and hyperlipidemia, key indices of human metabolic disease, with quantitative metabolic models that can be extrapolated to the mammalian system; and provide a systematic understanding of the metabolic and population processes shaping the efficacy of Lactobacillus (widely used as probiotic in human foods) for metabolic health. It will also provide a Drosophila model for probiotic research that can be extended beyond study of the impacts of interactions with gut microbiotas of different composition in this project to address other critically-important variables, including host genotype and diet.